qPCR - finally! (Week 7)

Each week I've been looking forward to completing the qPCR of the cave samples, but each week, it has been delayed for one reason or another. Last week, I was finally able to do it. The qPCR is a way to amplify DNA samples and figure out how many copies of DNA were in the tube before the amplification began. It's fun to use, because it creates real-time graphs showing the amount of DNA present after each cycle. The lines on the graph start off horizontal as the PCR begins and the amplification is slow. Then, they begin to rise sharply in a curve and, finally, level off. By comparing these amplification curves to the standard curves, the machine will calculate the number of copies of DNA that were added to each tube in the beginning.

From this qPCR data, I calculated the number of bacteria in each cave. Actually, the numbers I came up with are really just estimates, because there is a lot of room for error. For instance, each bacteria has a different number of copies of DNA, so I had to use an average. Also, I didn't necessarily get all the DNA from each soil sample during the extraction. Etc... However, this data gave us a nice comparison between the caves.

This week I was struggling with one of the new bacterial primers, which just didn't work. The other primers weren't ideal, but at least they had all worked when they were used with a strong positive control. However, one of the reverse bacterial primers just wouldn't work. This was troubling, because Rick and I were trying to figure out which bar-coded primers to buy and how much to buy. Finally, Rick figured out the problem: the primer was the wrong sequence. It  was the complementary sequence to the sequence we needed! It was nice to figure that out.

I'm on track to finish up the project in the next three weeks. I still can't believe how fast it is all going.